Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-11733234-B2
Application number	US-201715648339-A
Country	US
Kind code	B2
Filing date	Jul 12, 2017
Priority date	Jul 12, 2016
Publication date	Aug 22, 2023
Grant date	Aug 22, 2023

Abstract

Compositions, devices and methods are described for improving adhesion, attachment, and/or differentiation of cells in a microfluidic device or chip. In one embodiment, one or more ECM proteins are covalently coupled to the surface of a microchannel of a microfluidic device. The microfluidic devices can be stored or used immediately for culture and/or support of living cells such as mammalian cells, and/or for simulating a function of a tissue, e.g., a liver tissue, muscle tissue, etc. Extended adhesion and viability with sustained function over time is observed.

First claim

We claim:

1. A method of culturing cells, comprising:
a) providing a microfluidic device, comprising;
i) a microchannel comprising a polydimethylsiloxane porous membrane surface;
ii) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel;
iii) a source of ultraviolet light; and
iv) an N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker comprising an ultraviolet light reactive portion and a chemically reactive portion;

b) exposing said ultraviolet light reactive portion to said ultraviolet source;
c) covalently attaching one or more laminin proteins to said chemically reactive portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker;
d) seeding viable motor neuron cells on said one or more laminin proteins so as to create attached motor neuron cells;
e) flowing said fluid from said fluid source in a parallel orientation to said polydimethylsiloxane porous membrane surface so as to create flow conditions; and
f) culturing and differentiating said attached motor neuron cells under said flow conditions and said attached motor neuron cells remain attached for at least seven (7) days.

2. The method of claim 1 , wherein said attached motor neuron cells further remain functional for at least 7 days.

3. The method of claim 1 , wherein said attached motor neuron cells further remain functional for at least 14 days.

4. The method of claim 1 , wherein said:
i) exposing said ultraviolet light reactive portion comprises introducing said crosslinker or a solution containing said crosslinker to contact said polydimethylsiloxane porous membrane surface and permitting said crosslinker or said solution containing said crosslinker to react with said polydimethylsiloxane porous membrane surface during said ultraviolet light exposure; and
ii) covalently attaching said at least one laminin protein comprises introducing at least one laminin protein, or a solution containing at least one laminin protein to react with said crosslinker.

5. The method of claim 4 , wherein said introducing said crosslinker in i) further comprises exposing a selected area or pattern of said microchannel surface to said ultraviolet light source to create a selected area or pattern covalently attached to said ultraviolet light reactive portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker.

6. The method of claim 5 , wherein said exposing said selected area or pattern comprises masking said light so as to select said selected area or pattern.

7. The method of claim 5 , wherein said exposing said selected area or pattern comprises projecting said light in a light pattern so as to select said selected area or pattern.

8. The method of claim 5 , wherein said exposing said area or pattern comprises rastering light so as to select said selected area or pattern.

9. The method of claim 5 , wherein said selected area or pattern comprises a linear pattern.

10. A method of culturing cells, comprising:
a) providing a microfluidic device, comprising;
i) a microchannel comprising a polydimethylsiloxane patterned surface disposed within said microchannel, said microchannel is in fluidic communication with an intake port, at least one chamber and an outlet port;
ii) an N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)-hexanoate crosslinker comprising an ultraviolet light reactive portion and a chemically reactive portion;
iii) a source of ultraviolet light; and
iv) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel;

b) exposing said ultraviolet light reactive portion to said source of ultraviolet light;
c) covalently attaching one or more extracellular matrix proteins to said chemically reactive portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate crosslinker so as to create a treated patterned surface;
d) seeding viable epithelial cells on said extracellular matrix proteins so as to create attached viable epithelial cells;
e) flowing said fluid from said fluid source through said microchannel so as to create flow conditions; and
f) culturing said epithelial cells wherein said epithelial cells remain attached and viable for at least 7 days.

11. The method of claim 10 , wherein said attached epithelial cells further remain functional for at least 7 days.

12. The method of claim 10 , wherein said attached epithelial cells further remain functional for at least 14 days.

13. The method of claim 10 , wherein said patterned surface is a linear patterned surface.

14. The method of claim 10 , wherein said microfluidic device further comprises a membrane.

15. The method of claim 14 , wherein said membrane comprises said patterned surface.

16. A method of culturing cells, comprising:
a) providing a microfluidic device, comprising;
i) a microchannel comprising a polydimethylsiloxane surface disposed within said microchannel;
ii) an N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)-hexanoate crosslinker comprising an ultraviolet light reactive portion and a chemically reactive portion;
iii) a source of ultraviolet light; and
iv) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel;

b) masking a portion of said polymethylsiloxane surface to create a selected area or pattern;
c) irradiating said selected area or pattern with said source of ultraviolet light in the presence of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate crosslinker such that said ultraviolet light reactive portion covalently attaches to said selected area;
d) covalently attaching one or more extracellular matrix proteins to said chemically reactive portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate crosslinker;
e) putting said microfluidic device into storage;
f) seeding viable epithelial cells on said treated surface after storage so as to create attached epithelial cells;
g) flowing said fluid from said fluid source through said microchannel so as to create flow conditions; and
h) culturing said attached epithelial cells under said flow conditions such that and said attached epithelial cells remain attached and viable for at least 7 days.

17. The method of claim 16 , wherein said storage is a wet storage.

18. The method of claim 16 , wherein said storage is a dry storage.

19. The method of claim 16 , wherein said storage of said microfluidic device comprises a vapor proof packaging.

20. The method of claim 16 , wherein said polydimethylsiloxane surface is a linear patterned surface.

21. The method of claim 16 , wherein said microfluidic device further comprises a membrane.

22. The method of claim 21 , wherein said membrane comprises said polydimethylsiloxane surface.

23. A method of culturing cells, comprising:
a) providing a microfluidic device, comprising;
i) a microchannel comprising a polydimethylsiloxane selected area or pattern disposed within said microchannel;
ii) an N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker comprising an ultraviolet light reactive portion and a chemically reactive portion;
iii) a source of ultraviolet light; and
iv) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel;

b) covalently attaching one or more extracellular matrix protein

Assignees

• Emulate Inc

Inventors

• Levner Daniel
• Jang Kyung Jin
• Fraser Jacob
• Kerns S Jordan
• Varone Antonio
• Huh Dongeun

Classifications

• G01N33/5032 Primary

  on intercellular interactions · CPC title

• C07K2/00

  Peptides of undefined number of amino acids; Derivatives thereof · CPC title

• C07K14/78

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C12M23/16 Primary

  Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093) · CPC title

• C12M23/20

  Material Coatings (immunocoatings C12M25/00) · CPC title